Iron-sulfur clusters are present in more than 160 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of iron-sulfur biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged, termed the NIF, ISC and SUF systems, and in each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular-level understanding of iron-sulfur cluster biosynthesis in the NIF, ISC and SUF systems. Elucidating the mechanism of Fe-S cluster biosynthesis is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site specific changes in the target enzymes and proteins, biochemical and enzymatic assays, x-ray crystallography, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, electronic absorption and magnetic circular dichroism, resonance Raman, Mossbauer and mass spectrometry) that can probe the nature and detailed properties of iron or iron-sulfur centers during cluster biosynthesis. The objectives are to establish the structure of cluster-bound forms and the molecular mechanism of cluster assembly and transfer for each the three known types of iron-sulfur cluster scaffold proteins, characterize the mechanism of regulation of iron-sulfur cluster biosynthesis, and identify hitherto in characterized scaffold proteins specific of the assembly of cubane [4Fe-4S] clusters. [unreadable] [unreadable]